Bnl is cleaved at a single endoproteolytic site. (A) Location of putative PCS1-3 in the Bnl backbone. #1–4: GFP insertion sites; *, point mutations generated at PCS1 (M1) and PCS2 (M2). (A′) In silico predictions of PC sites. Upper panel, Furin-specific; lower panel, for general PC; green line, SP cleavage site; red line, a set threshold above which the sequence is predicted to be a PC site. (B–G) Examples of αHA immunostained (red) S2 cells expressing Bnl:GFP₃HA₄ (B) and Bnl:HA₁GFP₃ mutants as indicated (C–G); XYZ section near coverslip (D) and XZY section (E) of M1-expressing adherent cells. (H) Graphs comparing colocalization index (I_corr) of the HA- and GFP-tagged parts of Bnl:HA₁GFP₃ (WT), M1, M2, and M1M2 in αHA-stained (red) S2 cells. n = 15 (WT), 13 (M1), and 9 (M2 and M1M2); P values: ANOVA followed by Tukey HSD. In B–G, S2 cells were cotransfected with act-Gal4 and UAS-X, X= constructs as indicated. (I–M) Maximum-intensity projections of the wing-disc source (I′″, I″″, K, and M) expressing Bnl:HA₁GFP₃ mutants as indicated (bnl-Gal4 x UAS-X, X = M1, M2, M1M2) and the recipient ASPs (I–I″, J–J″, and L–L″). Blue, αDlg; white dashed line, ASP. In B–M, arrow, truncated Bnl:GFP₃ derivative; arrowhead, uncleaved Bnl:HA₁GFP₃. Scale bars: 10 µm (B–G); 30 µm (I–M).